Journal of Agricultural Science; Vol. 10, No. 5; 2018 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education Pathogenicity and Genetic Diversity of Fusarium oxysporum Causing Soybean Root Rot in Northeast China Yonggang Li1,2, Tongxue Zhao1, Gia Khuong Hoang Hua2, Lankun Xu1, Jinxin Liu1, Shuxian Li3, Hanwen Huang4 & Pingsheng Ji2 1 Agricultural College, Northeast Agricultural University, China 2 Department of Plant Pathology, University of Georgia, Tifton, GA, USA 3 USDA, Agricultural Research Service, Crop Genetics Research Unit, Stoneville, MS, USA 4 Department of Epidemiology and Biostatistics, University of Georgia, Athens, GA, USA Correspondence: Yonggang Li, Key Laboratory of Cold Crop Breeding Improvement and Physiological Ecology in Heilongjiang Province, Agricultural College, Northeast Agricultural University, Harbin 150030, China. Tel: 086-0451-55191064. E-mail: [email protected] Received: January 28, 2018 Accepted: March 11, 2018 Online Published: April 15, 2018 doi:10.5539/jas.v10n5p13 URL: https://doi.org/10.5539/jas.v10n5p13 Abstract Soybean is an important edible legume cultivated around the world. However, soybean production is seriously impacted by the widespread of root rot disease. In this study, genetic diversity and pathogenicity of Fusarium oxysporum associated with root rot of soybean in Heilongjiang province, China, were examined. A total of 50 F. oxysporum strains were isolated from diseased soybean plants grown in Harbin, Heihe, Jixi, Jiamusi and Qiqihar of Heilongjiang province. Pathogenicity study indicated that all F. oxysporum strains were able to induce root rot disease on soybean in which 28% of the isolates were highly aggressive, 42% were moderately aggressive, and 30% were weakly aggressive. Aggressiveness of the isolates did not appear to be associated with geographic location or plant age of isolation. Genomic DNA of the isolates was analyzed by polymerase chain reaction using eight amplified fragment length polymorphism (AFLP) primers that generated 1728 bands, of which 99% were polymorphic. Cluster analysis using UPGMA showed that the similarity values ranged from 0.15 to 0.47. At a similarity coefficient of 0.2, the isolates were separated into 7 groups. Analysis of molecular variance indicated that about 92% of the genetic variation resided within populations. No correlation was found between genetic diversity and aggressiveness or the geographic origin of the isolates. Results of the study indicate that pathogenic F. oxysporum are commonly associated with root rot of soybean with various aggressiveness and they are genetically diverse. Keywords: AFLP, soil borne pathogen, virulence, correlation 1. Introduction Soybean [Glycine max (L.) Merr.] is an important oilseed crop and a valuable source of vegetable proteins (Loganathan et al., 2010). Approximately 7 million tons of soybean seeds were produced in 69 countries in the world (FAO, 2016). With the production of 693 thousand tons of soybean seeds, China is considered as one of the world’s leading soybean producers (FAO, 2016). Total areas planted to soybean in China have been increasing, and the largest soybean producing province in the country is Heilongjiang (Li et al., 2013; Shurtleff & Aoyagi, 2016). Heilongjiang is located in northeast China with 4.3 million hectares of soybean produced in the province (Shurtleff & Aoyagi, 2016). Soybean production is hampered by the occurrence of root rot disease caused by Fusarium spp. Fusarium root rot has been a problem in soybean production in many countries worldwide (Sinclair & Backmen, 1989; Arias et al., 2013; Zhang et al., 2013a). Tap and lateral roots infected by the disease often become reddish brown or light to dark brown. The roots become shallow and fibrous and eventually rotted, and plants may be wilted especially under conditions of low moisture and high temperature. Root rot of soybean can be induced by various Fusarium species, with F. o x ys po ru m being the most common species reported (Nelson, 1999; Shiraishi et al., 2012; Zhang et al., 2013a). 13 jas.ccsenet.org Journal of Agricultural Science Vol. 10, No. 5; 2018 Management of Fusarium root rot of soybean is difficult. Information regarding fungicides effective for suppressing F. oxysporum on soybean is limited. In northeast China, growers often abandon production of soybean when the fields get heavy infestation by the pathogen due to lack of effective disease control measures and panic of severe yield loss. Host resistance is a recommended strategy for managing soil borne diseases. In a study conducted in Canada (Zhang et al., 2013a), 70 soybean cultivars were evaluated under field conditions and 17 cultivars with the lowest severity of root rot caused by F. oxysporum, ranging from 1.3 to 2.2, were the most resistant. Soybean cultivars with resistance to Fusarium root rot under conditions in northeast China are not known to be available. It is unknown neither if F. oxysporum causing root rot of soybean are phenotypically and genetically diverse. It is highly desirable to determine diversity of the pathogen for developing effective disease management programs such as the use of host resistance. Genomic fingerprinting techniques have been widely used to study genetic variability, population structure, and species phylogeny of F. oxysporum (Silva et al., 2013; Chen et al., 2014; Zimmermann et al., 2015). Among the techniques, amplified fragment length polymorphism (AFLP) is well recognized which has great discriminatory power (Vos et al., 1995; Mueller & Wolfenbarger, 1999; Silva et al., 2013). In a study on Fusarium wilt of bitter gourd caused by F. oxysporum f. sp. momordicae, AFLP analysis differentiated isolates with high virulence from those with low virulence, and pathogenicity of the 48 pathogen isolates was correlated with geographical locations of isolation of the isolates (Chen et al., 2014). In another study on F. oxysporum f. sp. radicis-cucumerinum from cucumbers, analysis with AFLP markers divided the 30 isolates in two distinct clusters (Tok & Kurt, 2010). All isolates in one cluster belonged to a vegetative compatibility group and isolates in the other cluster belonged to another vegetative compatibility group. These studies indicate that AFLP analysis is a useful tool in assessing genetic diversity of F. oxysporum populations. The objective of this study was to determine pathogenicity of F. oxysporum associated with root rot of soybean in northeast China and use AFLP technology to analyze genetic diversity of the pathogen. This study advances our understanding of the etiology of the disease and population genetics of the pathogen, which provides valuable information for developing disease management programs. 2. Materials and Methods 2.1 Sample Collection and Pathogen Identification Soybean plants showing root rot symptoms were sampled from different regions in Heilongjiang, China, in 2012 (Figure 1). Roots were washed with tap water, cut into pieces of 0.5 cm3, disinfested with 70% ethanol for 2 s and 0.5% NaOCl for 5 min, and rinsed three times with sterile distilled water. Root tissues were placed on potato dextrose agar (PDA) and incubated at 26 °C. Fungal hyphae grown from the tissues were transferred to fresh PDA plates. The isolates were identified as F. oxysporum according to cultural and morphological characteristics (Nelson et al., 1983; Leslie & Summerell, 2006). Single conidium isolates were obtained using the methods reported previously (Leslie & Summerell, 2006; Petkar et al., 2017). For molecular identification, mycelia from 5-day-old cultures on PDA were used to extract DNA using the methods reported by Zhang et al. (2010) with minor modifications. In brief, mycelia of the isolates were pulverized using liquid nitrogen and homogenized using extraction buffer containing cetyl trimethyl ammonium bromide (CTAB), 0.5% charcoal along with 0.2% β-mercaptoethanol. After incubation at 65 °C for 15 min, homogenates were purified three times with chloroform:isoamyl alcohol (24:1). The upper aqueous phase (400 μL) was transferred to a tube containing 800 μL isopropanol, and DNA pellets were obtained by adding 0.67 volumes of propanol. The pellets were washed with ice-cold ethanol (70%), air dried, dissolved in 50 μL of deionized water, and stored at 4 °C. Polymerase chain reaction (PCR) analysis was performed with F. oxysporum species-specific primers FOF1 and FOR1 using the conditions described previously (Zhang et al., 2013b). PCR products were visualized by running 1.5% agarose gel and observed under a gel documentation system. 14 jas.ccsenet.org Journal of Agricultural Science Vol. 10, No. 5; 2018 Figure 1. Map showing sampling locations of Fusarium oxysporum in Heilongjiang province, China 2.2 Pathogenicity Tests To prepare inoculum of the isolates, flasks containing 125 g of sorghum seeds and 50 ml of distilled water were autoclaved at 121 °C for 40 min in two consecutive days. A mycelial plug (7 mm diameter) taken from 7-day-old cultures of the isolates was transferred to each flask. The flasks were incubated aat 26 °C for a week and mixed by hand every 3 days. Ten sorghum seeds fully colonized by F. oxysporum were uniformly distributed on the top of vermiculite in a plastic pot (8 × 8 cm), and then covered with a 0.5 cm layer of sterile vermiculite. In the non-inoculated control, equal numbers of sterilized sorghum seeds were used. Twelve soybean seeds (cv. Hefeng 25) were surface-disinfested with 1.5% NaOCl for 5 min, washeed three times with sterile distilled water, and sown in each pot. A randomized complete block design was useed with three replicates and two pots for each isolate in each replicate. The pots were kept in a greenhouse at 25±3 °C, and seedlings in each pot were thiinned to 10 after emergence. The seedlings were watered daily using overhead irrigation to maintain soil moisture. To evaluate disease severity, seedlings were removed from the pots 10 days after emergence and roots were washed under running tap water. Disease was rated using a 0-to-7 scale previously described by Li et al.